Undamaged DNA Research Articles

Structural basis for recognition of distinct deaminated DNA lesions by endonuclease Q

Significance Deamination of cytosine and adenine bases in DNA is one of the most common sources of genetic mutation in biology and a major driver of virus and tumor evolution. Recent studies have identified a novel class of DNA-repair enzyme from archaea and bacteria, designated endonuclease Q (EndoQ), which has unique activity to initiate the repair of both these deaminated lesions. Our X-ray crystallographic and biochemical analyses presented here reveal how EndoQ achieves high selectivity toward these chemically divergent substrates without cleaving undamaged DNA. We also demonstrate that EndoQ, as the only known nuclease that cleaves DNA at deoxyuridine nucleotide, is useful for studying DNA deamination and repair in mammalian systems.

Tracking Escherichia coli DNA polymerase V to the entire genome during the SOS response

Ribonucleotides are frequently incorporated into DNA and can be used as a marker of DNA replication enzymology. To investigate on a genome-wide scale, how E. coli pol V accesses undamaged chromosomal DNA during the SOS response, we mapped the location of ribonucleotides incorporated by steric gate variants of pol V across the entire E. coli genome. To do so, we used strains that are deficient in ribonucleotide excision repair (ΔrnhB), deficient in pol IV DNA polymerase, constitutively express all SOS-regulated genes [lexA(Def)] and constitutively “activated” RecA* (recA730). The strains also harbor two steric gate variants of E. coli pol V (Y11A, or F10L), or a homolog of pol V, (pol VR391-Y13A). Ribonucleotides are frequently incorporated by the pol V-Y11A and pol VR391-Y13A variants, with a preference to the lagging strand. In contrast, the pol V-F10L variant incorporates less ribonucleotides and no strand preference is observed. Sharp transitions in strand specificity are observed at the replication origin (oriC), while a gradient is observed at the termination region. To activate RecA* in a recA+ strain, we treated the strains with ciprofloxacin and genome-wide mapped the location of the incorporated ribonucleotides. Again, the pol V-Y11A steric gate variant exhibited a lagging strand preference. Our data are consistent with a specific role for pol V in lagging strand DNA synthesis across the entire E. coli genome during the SOS response.

The DNA ligands Arg47 and Arg76 are crucial for catalysis by human PrimPol

Human primase and DNA polymerase PrimPol re-starts stalled replication forks by repriming downstream DNA lesions and protects cells against DNA damage. Structure of the catalytic core of PrimPol with DNA primer, template and incoming dATP was solved but the mechanisms of DNA polymerase and primase activities of PrimPol are not fully understood. In this work, using site-directed mutagenesis we biochemically analyzed the role of active site residues Arg47 and Arg76 contacting DNA template in DNA polymerase and primase activities of PrimPol. The substitution R47A diminished the DNA polymerase and primase activities of PrimPol whereas the single amino acid substitution R76A caused almost complete loss of catalytic activities. Both amino acid substitutions affected the spectrum of dNMPs incorporation on undamaged DNA templates and opposite 8-oxoguanine. Finally, substitutions of the Arg47 and Arg76 residues attenuated the formation of the stable PrimPol:DNA complex in the presence of ATP/dNTPs. Together, these findings suggest a key role of the Arg47 and Arg76 in DNA synthesis by PrimPol.

Functional and Flow Cytometric Analysis of Buffalo Cryopreserved Spermatozoa: Comparison of Different Breeds and Incubation Times.

Background:The purpose of this research was to compare the functional parameters of frozen-thawed Iranian Azari buffalo spermatozoa with imported semen samples of Italian Mediterranean buffalo (IMB) after the thawing process and 4 hours of incubation.Materials and Methods:In this experimental study, a total of twenty-four ejaculates from four Iranian Azari buffalo bulls were collected. Semen samples were diluted in AndroMed extender at a concentration of 50×106 spermatozoa/ ml. The diluted samples were filled in 0.5 ml straws and were frozen in a programmable freezer. For imported semen samples, twenty-four samples of four IMB were used, which were diluted in AndroMed extender and frozen by the same procedure. Frozen-thawed sperm motion patterns, mitochondrial activity, membrane integrity, DNA integrity, reactive oxygen species (ROS), and apoptosis status were evaluated immediately after thawing and 4 hours of incubation.Results:Post-thawed sperm motility, progressive motility (PM), mitochondrial activity, membrane integrity were significantly higher in imported semen samples in compare with Iranian Azari buffalo. After 4 hours of incubation, sperm velocity patterns were superior in Iranian Azari semen samples. Moreover, the percentage of sperm cells with un-damaged DNA was higher in Iranian semen samples compared to imported samples at the time 0 of incubation. Following 4 hours of incubation, a significant increase in intracellular ROS level leads to reduced membrane integrity, mitochondrial activity, and DNA integrity in both buffalo breeds. At time 4, Iranian samples showed significantly lower apoptosis and higher dead spermatozoa compared to imported semen samples.Conclusion:Our study showed that the post-thawed quality of Iranian Azari buffalo semen was comparable with imported samples after 4 hours of incubation. Further investigations are recommended to assess the in vitro and in vivo fertility rate of both buffalo breeds.

Selective Metal Ion Utilization Contributes to the Transformation of the Activity of Yeast Polymerase η from DNA Polymerization toward RNA Polymerization.

Polymerase eta (Polη) is a translesion synthesis DNA polymerase directly linked to cancer development. It can bypass several DNA lesions thereby rescuing DNA damage-stalled replication complexes. We previously presented evidence implicating Saccharomyces cerevisiae Polη in transcription elongation, and identified its specific RNA extension and translesion RNA synthetic activities. However, RNA synthesis by Polη proved rather inefficient under conditions optimal for DNA synthesis. Searching for factors that could enhance its RNA synthetic activity, we have identified the divalent cation of manganese. Here, we show that manganese triggers drastic changes in the activity of Polη. Kinetics experiments indicate that manganese increases the efficiency of ribonucleoside incorporation into RNA by ~400–2000-fold opposite undamaged DNA, and ~3000 and ~6000-fold opposite TT dimer and 8oxoG, respectively. Importantly, preference for the correct base is maintained with manganese during RNA synthesis. In contrast, activity is strongly impaired, and base discrimination is almost lost during DNA synthesis by Polη with manganese. Moreover, Polη shows strong preference for manganese during RNA synthesis even at a 25-fold excess magnesium concentration. Based on this, we suggest that a new regulatory mechanism, selective metal cofactor utilization, modulates the specificity of Polη helping it to perform distinct activities needed for its separate functions during replication and transcription.

Tethering-facilitated DNA 'opening' and complementary roles of β-hairpin motifs in the Rad4/XPC DNA damage sensor protein.

XPC/Rad4 initiates eukaryotic nucleotide excision repair on structurally diverse helix-destabilizing/distorting DNA lesions by selectively ‘opening’ these sites while rapidly diffusing along undamaged DNA. Previous structural studies showed that Rad4, when tethered to DNA, could also open undamaged DNA, suggesting a ‘kinetic gating’ mechanism whereby lesion discrimination relied on efficient opening versus diffusion. However, solution studies in support of such a mechanism were lacking and how ‘opening’ is brought about remained unclear. Here, we present crystal structures and fluorescence-based conformational analyses on tethered complexes, showing that Rad4 can indeed ‘open’ undamaged DNA in solution and that such ‘opening’ can largely occur without one or the other of the β-hairpin motifs in the BHD2 or BHD3 domains. Notably, the Rad4-bound ‘open’ DNA adopts multiple conformations in solution notwithstanding the DNA’s original structure or the β-hairpins. Molecular dynamics simulations reveal compensatory roles of the β-hairpins, which may render robustness in dealing with and opening diverse lesions. Our study showcases how fluorescence-based studies can be used to obtain information complementary to ensemble structural studies. The tethering-facilitated DNA ‘opening’ of undamaged sites and the dynamic nature of ‘open’ DNA may shed light on how the protein functions within and beyond nucleotide excision repair in cells.

The role of active-site amino acid residues in the cleavage of DNA and RNA substrates by human apurinic/apyrimidinic endonuclease APE1

BackgroundHuman apurinic/apyrimidinic endonuclease APE1 is one of participants of the DNA base excision repair pathway. APE1 processes AP-sites and many other types of DNA damage via hydrolysis of the phosphodiester bond on the 5′ side of the lesion. APE1 also acts as an endoribonuclease, i.e., can cleave undamaged RNA. MethodsUsing pre-steady-state kinetic analysis we examined the role of certain catalytically important amino acids in APE1 enzymatic pathway and described their involvement in the mechanism of the target nucleotide recognition. ResultsComparative analysis of the cleavage efficiency of damaged DNAs containing an abasic site, 5,6-dihydrouridine, or α-anomer of adenosine as well as 3′-5′-exonuclease degradation of undamaged DNA and endonuclease hydrolysis of RNA substrates by mutant APE1 enzymes containing a substitution of an active-site amino acid residue (D210N, N212A, T268D, M270A, or D308A) was performed. Detailed pre–steady-state kinetics of conformational changes of the enzyme and of DNA substrate molecules during recognition and cleavage of the abasic site were studied. ConclusionsIt was revealed that substitution T268D significantly disturbed initial DNA binding, whereas Asn212 is critical for the DNA-bending stage and catalysis. Substitution D210N increased the binding efficacy and blocked the catalytic reaction, but D308A decreased the binding efficacy owing to disruption of Mg2+ coordination. Finally, the substitution of Met270 also destabilized the enzyme–substrate complex but did not affect the catalytic reaction. SignificanceIt was found that the tested substitutions of the active-site amino acid residues affected different stages of the complex formation process as well as the catalytic reaction.

Impact of the Nucleosome Histone Core on the Structure and Dynamics of DNA-Containing Pyrimidine-Pyrimidone (6-4) Photoproduct.

The pyrimidine-pyrimidone (6-4) photoproduct (64-PP) is an important photoinduced DNA lesion constituting a mutational signature for melanoma. The structural impact of 64-PP on DNA complexed with histones affects the lesion mutagenicity and repair but remains poorly understood. Here we investigate the conformational dynamics of DNA-containing 64-PP within the nucleosome core particle by atomic-resolution molecular dynamics simulations and multiscale data analysis. We demonstrate that the histone core exerts important mechanical restraints that largely decrease global DNA structural fluctuations. However, the local DNA flexibility at the damaged site is enhanced due to imperfect structural adaptation to restraints imposed by the histone core. If 64-PP faces the histone core and is therefore not directly accessible by the repair protein, the complementary strand facing the solvent is deformed and exhibits higher flexibility than the corresponding strand in a naked, undamaged DNA. This may serve as an initial recognition signal for repair. Our simulations also pinpoint the structural role of proximal residues from the truncated histone tails.

Lysines in the lyase active site of DNA polymerase β destabilize nonspecific DNA binding, facilitating searching and DNA gap recognition

DNA polymerase (pol) β catalyzes two reactions at DNA gaps generated during base excision repair, gap-filling DNA synthesis and lyase-dependent 5´-end deoxyribose phosphate removal. The lyase domain of pol β has been proposed to function in DNA gap recognition and to facilitate DNA scanning during substrate search. However, the mechanisms and molecular interactions used by pol β for substrate search and recognition are not clear. To provide insight into this process, a comparison was made of the DNA binding affinities of WT pol β, pol λ, and pol μ, and several variants of pol β, for 1-nt-gap-containing and undamaged DNA. Surprisingly, this analysis revealed that mutation of three lysine residues in the lyase active site of pol β, 35, 68, and 72, to alanine (pol β KΔ3A) increased the binding affinity for nonspecific DNA ∼11-fold compared with that of the WT. WT pol μ, lacking homologous lysines, displayed nonspecific DNA binding behavior similar to that of pol β KΔ3A, in line with previous data demonstrating both enzymes were deficient in processive searching. In fluorescent microscopy experiments using mouse fibroblasts deficient in PARP-1, the ability of pol β KΔ3A to localize to sites of laser-induced DNA damage was strongly decreased compared with that of WT pol β. These data suggest that the three lysines in the lyase active site destabilize pol β when bound to DNA nonspecifically, promoting DNA scanning and providing binding specificity for gapped DNA.

Detection of OG:A Lesion Mispairs by MutY Relies on a Single His Residue and the 2-Amino Group of 8-Oxoguanine.

MutY glycosylase excises adenines misincorporated opposite the oxidatively damaged lesion, 8-oxo-7,8-dihydroguanine (OG), to initiate base excision repair and prevent G to T transversion mutations. Successful repair requires MutY recognition of the OG:A mispair amidst highly abundant and structurally similar undamaged DNA base pairs. Herein we use a combination of in vitro and bacterial cell repair assays with single-molecule fluorescence microscopy to demonstrate that both a C-terminal domain histidine residue and the 2-amino group of OG base are critical for MutY detection of OG:A sites. These studies are the first to directly link deficiencies in MutY lesion detection with incomplete cellular repair. These results suggest that defects in lesion detection of human MutY (MUTYH) variants may prove predictive of early-onset colorectal cancer known an MUTYH-associated polyposis. Furthermore, unveiling these specific molecular determinants for repair makes it possible to envision new MUTYH-specific cancer therapies.

Abstract IA20: Novel cell microarray technologies for studies of DNA damage and cell survival and applications for studies of microbe-induced DNA damage

Abstract Genotoxicity testing is critical for predicting adverse effects of pharmaceutical, industrial, and environmental chemicals. The comet assay is a commonly used approach for detecting DNA damage that is based on the underlying principle that damaged DNA migrates more readily than undamaged DNA when electrophoresed in agarose. We previously developed a higher-throughput version of the comet assay (Wood DK, PNAS 2010). For the “CometChip” assay, cells are loaded into an array of agarose microwells to create a cell microarray. The CometChip can be fully automated, making the assay more than 1000X faster, while improving reproducibility. While effective for detecting single-strand breaks, abasic sites, and alkali-sensitive sites, the alkaline comet assay is not effective for detection of carcinogenic bulky DNA lesions that do not impact DNA mobility and that require metabolic activation. To overcome this, we use DNA replication inhibitors (hydroxyurea and 1-β-d-arabinofuranosyl cytosine) shown to trap single-strand breaks that are formed during nucleotide excision repair, which normally removes bulky lesions. Thus, comet-undetectable bulky lesions are converted into comet-detectable single-strand breaks. Together with HepaRG™ cells that recapitulate in vivo metabolic capacity, we have thus created the “HepaCometChip,” which provides a more broadly effective approach for detection of DNA damage. In addition to genotoxicity testing, the microwell array has also been harnessed for use in a cell survival assay. For the “MicroColonyChip” (Ngo LP, Cell Reports 2019), cells are loaded into the agarose microwell array and allowed to grow to form microcolonies. Differences in cell survival can be detected by changes in the distribution of colony sizes, a novel metric for cell survival quantitation. The assay is as sensitive as the traditional “gold standard” colony-forming assay, yet it takes days instead of weeks to complete. Parallel studies of genotoxicity and cytotoxicity can thus be performed using a shared microwell platform. These tools have broad utility, including studies of microbe-induced DNA damage. Examples of S. pneumoniae and E. coli microbial product-induced double-strand breaks and interstrand crosslinks contribute to an emerging paradigm wherein pathogen-induced DNA damage has the potential to promote disease. Citation Format: Le P. Ngo, Prashant Rai, Matthew R. Wilson, Carole Swartz, John Winters, Yang Su, Jing Ge, Tze-Khee Chan, Emily P. Balskus, Vincent Chow, Les Recio, Leona D. Samson, Bevin P. Engelward. Novel cell microarray technologies for studies of DNA damage and cell survival and applications for studies of microbe-induced DNA damage [abstract]. In: Proceedings of the AACR Special Conference on Environmental Carcinogenesis: Potential Pathway to Cancer Prevention; 2019 Jun 22-24; Charlotte, NC. Philadelphia (PA): AACR; Can Prev Res 2020;13(7 Suppl): Abstract nr IA20.

Abstract B042: Loss of nuclear alpha catenin contributes to chemoresistance and aggressive disease in African American triple-negative breast cancer patients

Abstract Triple-negative breast cancer (TNBC) is a particularly aggressive, difficult-to-treat subtype of the disease. A well-documented health disparity exists within TNBC: African American (AA) women are more likely to be diagnosed with and die from the disease. Our group reported homozygous deletions in the CTNNA1 gene, which encodes the protein alpha catenin, in AA TNBC. We have undertaken a basic and translational research study to understand the mechanistic role and clinical impact of alpha catenin loss in TNBC, particularly in AA patients. To validate our findings of alpha catenin loss in TNBC, we analyzed over 600 breast cancer patient samples by immunohistochemistry. We found that alpha catenin protein loss was inversely correlated with survival in TNBC. Moreover, loss was observed more frequently in tumors from AA patients. Interestingly, both nuclear and cytosolic alpha catenin adhered to this pattern. While its cytosolic role has been well studied, little is known about nuclear alpha catenin and its role in disease. To understand the nuclear function of alpha catenin, we developed several isogenic cell line pairs. Using CRISPR/Cas9-mediated gene editing, we generated CTNNA1 knockout (KO) BT-549 and MB-MDA-436 cell lines. We also reintroduced CTNNA1 into MDA-MB-468 cells—a line derived from an AA woman with an endogenous deletion in CTNNA1. Using these models, we found that alpha catenin KO cells demonstrated greater capacity for anchorage-independent growth. Loss of alpha catenin also resulted in decreased sensitivity to DNA-damaging chemotherapies including platinum salts (cisplatin, carboplatin), topoisomerase poisons (doxorubicin, etoposide), and the PARP inhibitor olaparib. This is clinically relevant as doxorubicin, carboplatin, and olaparib are approved for treatment of TNBC patients. To identify binding partners, we performed co-immunoprecipitation of alpha catenin from nuclear lysates, followed by mass spectrometry. We found that nuclear alpha catenin can interact directly with ATR, a kinase critical to the DNA damage response (DDR). ATR mediates both the repair of DNA lesions and the G2/M cell cycle checkpoint, which ensures that only cells with undamaged DNA may enter mitosis. We further found that alpha catenin KO cells were more sensitive to inhibitors of ATR, as well as to inhibitors of the G2/M checkpoint proteins Chk1 and Wee1. Tellingly, the KO cells were less sensitive to inhibitors of ATM or DNA-PK, two regulators of alternate DDR pathways. This suggests that nuclear alpha catenin likely plays a specific role in ATR-directed processes. In our study, we have identified nuclear alpha catenin as a tumor suppressor that affects TNBC's susceptibility to chemotherapy by playing a role in the DDR and the G2/M checkpoint. Our data suggest that loss of alpha catenin is more common in AA patients, and is associated with poor prognosis. Therefore, CTNNA1 status may be important in determining appropriate therapeutic strategies in a subset of patients. Citation Format: Rania Bassiouni, Victoria Dirgo, Krystine Mansfield, Ritin Sharma, Lee D. Gibbs, Patrick Pirrotte, Nasreen Vohra, Kevin Gardner, John D. Carpten. Loss of nuclear alpha catenin contributes to chemoresistance and aggressive disease in African American triple-negative breast cancer patients [abstract]. In: Proceedings of the Eleventh AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2018 Nov 2-5; New Orleans, LA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl):Abstract nr B042.

The Potential Use of Rice Husk for Reducing the Genotoxic Effects of Iron and Aluminum Oxides Nanoparticles in Oreochromis niloticus

Metal oxide nanoparticles have wide applications, which have elevated serious alarms about their impacts on the environment. Therefore, we investigated the potential adsorptive capacity of rice husk toward Fe2O3 and Al2O3 nanoparticles to reduce their genotoxic effects. Fish were subjected to 10 mg/l of Fe2O3 and Al2O3 nanoparticles in single and combined doses with and without rice husk water treatment for 7 days. The genotoxic effects were evaluated using the micronucleus test in the peripheral blood and comet assay in liver tissues. Significant elevation of micronuclei induction in addition to eight nuclear and cytoplasmic abnormalities (P < 0.05) was observed in all fish groups compared to the control groups. Fish that exposed to Fe2O3 nanoparticle showed the maximum induction of all recorded anomalies. Moreover, two indices of DNA damage were evaluated by the comet assay (comet score and % tail DNA) in liver tissues. The scoring of comet cells indicated that the highest frequencies of stage 0 (undamaged DNA) were in control and Al2O3 exposed groups, while stage 4 (extensive DNA damage) was significantly elevated in Fe2O3 exposed fish. The % of DNA damage was maximized in the Fe2O3 nanoparticles exposed fish and minimized in Al2O3 nanoparticles exposed fish. Based on the frequencies of nuclear anomalies, degree, and percentage of DNA damage, all rice husk treated groups showed a marked reduction in the genotoxic damage compared with untreated groups. Finally, both nanoparticles showed genotoxic potential and the rice husk had an efficient absorptive capacity for both of them individually or combined.

Recognition of 1,N2-ethenoguanine by alkyladenine DNA glycosylase is restricted by a conserved active-site residue

The adenine, cytosine, and guanine bases of DNA are susceptible to alkylation by the aldehyde products of lipid peroxidation and by the metabolic byproducts of vinyl chloride pollutants. The resulting adducts spontaneously cyclize to form harmful etheno lesions. Cells employ a variety of DNA repair pathways to protect themselves from these pro-mutagenic modifications. Human alkyladenine DNA glycosylase (AAG) is thought to initiate base excision repair of both 1,N6-ethenoadenine (ϵA) and 1,N2-ethenoguanine (ϵG). However, it is not clear how AAG might accommodate ϵG in an active site that is complementary to ϵA. This prompted a thorough investigation of AAG-catalyzed excision of ϵG from several relevant contexts. Using single-turnover and multiple-turnover kinetic analyses, we found that ϵG in its natural ϵG·C context is very poorly recognized relative to ϵA·T. Bulged and mispaired ϵG contexts, which can form during DNA replication, were similarly poor substrates for AAG. Furthermore, AAG could not recognize an ϵG site in competition with excess undamaged DNA sites. Guided by previous structural studies, we hypothesized that Asn-169, a conserved residue in the AAG active-site pocket, contributes to discrimination against ϵG. Consistent with this model, the N169S variant of AAG was 7-fold more active for excision of ϵG compared with the wildtype (WT) enzyme. Taken together, these findings suggest that ϵG is not a primary substrate of AAG, and that current models for etheno lesion repair in humans should be revised. We propose that other repair and tolerance mechanisms operate in the case of ϵG lesions.

New structural insights into the recognition of undamaged splayed-arm DNA with a single pair of non-complementary nucleotides by human nucleotide excision repair protein XPA

XPA (Xeroderma pigmentosum complementation group A) is a core scaffold protein that plays significant roles in DNA damage verification and recruiting downstream endonucleases in the nucleotide excision repair (NER) pathway. Here, we present the 2.81 Å resolution crystal structure of the DNA-binding domain (DBD) of human XPA in complex with an undamaged splayed-arm DNA substrate with a single pair of non-complementary nucleotides. The structure reveals that two XPA molecules bind to one splayed-arm DNA with a 10-bp duplex recognition motif in a non-sequence-specific manner. XPA molecules bind to both ends of the DNA duplex region with a characteristic β-hairpin. A conserved tryptophan residue Trp175 packs against the last base pair of DNA duplex and stabilizes the conformation of the characteristic β-hairpin. Upon DNA binding, the C-terminal last helix of XPA would shift towards the minor groove of the DNA substrate for better interaction. Notably, human XPA is able to bind to the undamaged DNA duplex without any kinks, and XPA-DNA binding does not bend the DNA substrate obviously. This study provides structural basis for the binding mechanism of XPA to the undamaged splayed-arm DNA with a single pair of non-complementary nucleotides.

Single-molecule visualization reveals the damage search mechanism for the human NER protein XPC-RAD23B.

DNA repair is critical for maintaining genomic integrity. Finding DNA lesions initiates the entire repair process. In human nucleotide excision repair (NER), XPC-RAD23B recognizes DNA lesions and recruits downstream factors. Although previous studies revealed the molecular features of damage identification by the yeast orthologs Rad4-Rad23, the dynamic mechanisms by which human XPC-RAD23B recognizes DNA defects have remained elusive. Here, we directly visualized the motion of XPC-RAD23B on undamaged and lesion-containing DNA using high-throughput single-molecule imaging. We observed three types of one-dimensional motion of XPC-RAD23B along DNA: diffusive, immobile and constrained. We found that consecutive AT-tracks led to increase in proteins with constrained motion. The diffusion coefficient dramatically increased according to ionic strength, suggesting that XPC-RAD23B diffuses along DNA via hopping, allowing XPC-RAD23B to bypass protein obstacles during the search for DNA damage. We also examined how XPC-RAD23B identifies cyclobutane pyrimidine dimers (CPDs) during diffusion. XPC-RAD23B makes futile attempts to bind to CPDs, consistent with low CPD recognition efficiency. Moreover, XPC-RAD23B binds CPDs in biphasic states, stable for lesion recognition and transient for lesion interrogation. Taken together, our results provide new insight into how XPC-RAD23B searches for DNA lesions in billions of base pairs in human genome.

Kinetic Investigation of Translesion Synthesis across a 3-Nitrobenzanthrone-Derived DNA Lesion Catalyzed by Human DNA Polymerase Kappa

3-Nitrobenzanthrone (3-NBA) is a byproduct of diesel exhaust and is highly present in industrial and populated areas. Inhalation of 3-NBA results in formation of N-(2'-deoxyguanosin-8-yl)-3-aminobenzanthrone (dGC8-N-ABA), a bulky DNA lesion that is of concern due to its mutagenic and carcinogenic potential. If dGC8-N-ABA is not bypassed during genomic replication, the lesion can stall cellular DNA replication machinery, leading to senescence or apoptosis. We have previously used running start assays to demonstrate that human DNA polymerases eta (hPolη) and kappa (hPolκ) are able to catalyze translesion DNA synthesis (TLS) across a site-specifically placed dGC8-N-ABA in a DNA template. Consistently, gene knockdown of hPolη and hPolκ in HEK293T cells reduces the efficiency of TLS across dGC8-N-ABA by ∼25 and ∼30%, respectively. Here, we kinetically investigated why hPolκ paused when bypassing and extending from dGC8-N-ABA. Our kinetic data show that correct dCTP incorporation efficiency of hPolκ dropped by 116-fold when opposite dGC8-N-ABA relative to undamaged dG, leading to hPolκ pausing at the lesion site observed in the running start assays. The already low nucleotide incorporation fidelity of hPolκ was further decreased by 10-fold during lesion bypass, and thus, incorrect nucleotides, especially dATP, were incorporated opposite dGC8-N-ABA with comparable efficiencies as correct dCTP. With regard to the dGC8-N-ABA bypass product extension step, hPolκ incorporated correct dGTP onto the damaged DNA substrate with a 786-fold lower efficiency than onto the corresponding undamaged DNA substrate, which resulted in hPolκ pausing at the site in the running start assays. Furthermore, hPolκ extended the primer-terminal matched base pair dC:dGC8-N-ABA with a 100-1000-fold lower fidelity than it extended the undamaged dC:dG base pair. Together, our kinetic results strongly indicate that hPolκ was error-prone during TLS of dGC8-N-ABA.

Abstract 3488: Loss of nuclear alpha-catenin is associated with race, aggressive disease, and chemoresistance in triple negative breast cancer

Abstract Triple negative breast cancer (TNBC) is a particularly aggressive and difficult-to-treat subtype of the disease. A known health disparity exists within TNBC: African American (AA) women are more likely to be diagnosed with and die from the disease. Our group previously reported homozygous deletions in the CTNNA1gene, which encodes the protein alpha-catenin, in AA TNBC. We have undertaken a basic and translational research study to understand the mechanistic role and clinical impact of alpha-catenin loss in TNBC. To validate our findings of alpha-catenin loss in TNBC, we analyzed over 500 breast cancer patient samples by immunohistochemistry. We found loss of both nuclear and cytoplasmic alpha-catenin to be inversely correlated with survival in TNBC. However, loss of nuclear alpha-catenin was observed more frequently in tumors from AA patients, and corresponded to poorer survival in this group. While its cytosolic role has been well studied, little is known about nuclear alpha-catenin and its role in disease. To examine the nuclear function of alpha-catenin, we used CRISPR/Cas9-mediated gene editing to generate CTNNA1 knockout (KO) BT-549 and MB-MDA-436 cell lines. We also reintroduced CTNNA1 into MDA-MB-468 cells - a line derived from an AA woman with an endogenous deletion in CTNNA1. Using these models, we confirmed that all lines contained a pool of nuclear alpha-catenin. To identify binding partners, we performed co-immunoprecipitation of alpha-catenin from nuclear lysates, followed by mass spectrometry. Nuclear alpha-catenin was found to interact directly with ATR, a kinase critical to the DNA damage response (DDR). ATR mediates both the repair of DNA lesions and the G2/M cell cycle checkpoint to ensure that only cells with undamaged DNA may enter mitosis. We found high levels of activated ATR in KO cells, which corresponded with more efficient DNA repair after exposure to UV light. Loss of alpha-catenin also resulted in decreased sensitivity to the DNA-damaging chemotherapeutics cisplatin, carboplatin, doxorubicin, etoposide, and olaparib, several of which are approved for treatment of TNBC patients. Furthermore, alpha-catenin KO cells were more sensitive to inhibitors of ATR, as well as to inhibitors of the G2/M checkpoint proteins Chk1 and Wee1. Tellingly, the KO cells were less sensitive to inhibitors of ATM or DNA-PK, two regulators of alternate DDR pathways. This suggests that nuclear alpha-catenin likely plays a specific role in ATR-directed processes. In our studies, we have identified nuclear alpha-catenin as a tumor suppressor that affects TNBC’s susceptibility to chemotherapy by playing a role in the DDR and the G2/M checkpoint. Our data suggest that loss of alpha-catenin is more common in AA patients, and is associated with poor prognosis. Therefore, CTNNA1 status may be important in determining appropriate therapeutic strategies for this subset of patients. Citation Format: Rania Bassiouni, Yunchi Li, Victoria David-Dirgo, Krystine Garcia-Mansfield, Ritin Sharma, Patrick Pirrotte, Nasreen Vohra, Sandeep Singhal, Kevin Gardner, John D. Carpten. Loss of nuclear alpha-catenin is associated with race, aggressive disease, and chemoresistance in triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3488.

RADAR-seq: A RAre DAmage and Repair sequencing method for detecting DNA damage on a genome-wide scale

RAre DAmage and Repair sequencing (RADAR-seq) is a highly adaptable sequencing method that enables the identification and detection of rare DNA damage events for a wide variety of DNA lesions at single-molecule resolution on a genome-wide scale. In RADAR-seq, DNA lesions are replaced with a patch of modified bases that can be directly detected by Pacific Biosciences Single Molecule Real-Time (SMRT) sequencing. RADAR-seq enables dynamic detection over a wide range of DNA damage frequencies, including low physiological levels. Furthermore, without the need for DNA amplification and enrichment steps, RADAR-seq provides sequencing coverage of damaged and undamaged DNA across an entire genome. Here, we use RADAR-seq to measure the frequency and map the location of ribonucleotides in wild-type and RNaseH2-deficient E. coli and Thermococcus kodakarensis strains. Additionally, by tracking ribonucleotides incorporated during in vivo lagging strand DNA synthesis, we determined the replication initiation point in E. coli, and its relation to the origin of replication (oriC). RADAR-seq was also used to map cyclobutane pyrimidine dimers (CPDs) in Escherichia coli (E. coli) genomic DNA exposed to UV-radiation. On a broader scale, RADAR-seq can be applied to understand formation and repair of DNA damage, the correlation between DNA damage and disease initiation and progression, and complex biological pathways, including DNA replication.

A Drosophila cell-free system that senses DNA breaks and triggers phosphorylation signalling.

Preblastoderm Drosophila embryo development is characterized by fast cycles of nuclear divisions. Extracts from these embryos can be used to reconstitute complex chromatin with high efficiency. We now discovered that this chromatin assembly system contains activities that recognize unprotected DNA ends and signal DNA damage through phosphorylation. DNA ends are initially bound by Ku and MRN complexes. Within minutes, the phosphorylation of H2A.V (homologous to γH2A.X) initiates from DNA breaks and spreads over tens of thousands DNA base pairs. The γH2A.V phosphorylation remains tightly associated with the damaged DNA and does not spread to undamaged DNA in the same reaction. This first observation of long-range γH2A.X spreading along damaged chromatin in an in vitro system provides a unique opportunity for mechanistic dissection. Upon further incubation, DNA ends are rendered single-stranded and bound by the RPA complex. Phosphoproteome analyses reveal damage-dependent phosphorylation of numerous DNA-end-associated proteins including Ku70, RPA2, CHRAC16, the exonuclease Rrp1 and the telomer capping complex. Phosphorylation of spindle assembly checkpoint components and of microtubule-associated proteins required for centrosome integrity suggests this cell-free system recapitulates processes involved in the regulated elimination of fatally damaged syncytial nuclei.